Cell calcification supressing proteins, and genes of the proteins

ABSTRACT

This invention provides cell-calcification inhibitory proteins as well as genes encoding the proteins. Based on the discovery of a novel isoform gene of the c-erg gene (herein referred to as “C-11 gene”) which is an erg gene derived from chickens, the nucleotide sequence of the gene has been determined, and then the expression of a protein encoded by such gene (herein referred to as “C-11 protein”) has been confirmed. Further, it has been discovered that when the c-erg or C-11 gene is introduced into osteoblasts, the calcification of the cells is inhibited.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to cell-calcification inhibitory proteins as wellas to genes encoding such proteins.

2. Description of Related Art

Ets genes were first identified as oncogene of avian acute leukemiavirus E26. Recently, the family of Ets related genes (Ets genesuperfamily) has been found in a host ranging from human to Drosophila.It is believed that these genes are a transcriptional modulator whichplays a basic, important role in the control of proliferation anddifferentiation of cells. In contrast, the biological functions of theEts gene superfamily are hardly known.

SUMMARY OF THE INVENTION

Recently, it has been reported that the Ets related genes (erg) arespecifically expressed at the sites of cartilage formation. Thisindicated the possibility of erg's being involved to some extent inskeleton formation at its initial stages. The present inventors, basedon such findings, have accomplished the invention by introducing intoosteoblasts, an erg gene derived from chickens (herein referred to as“chicken-erg gene” or “c-erg gene”) and by successfully elucidating thefunctions of the c-erg gene.

BRIEF DESCRIPTION OF THE DRAWINGS

This application contains at least one figure in color. Copies of apatent issuing from the application, which includes color figures, willbe available from the United States Patent and Trademark Office uponrequest and payment of necessary fees.

FIG. 1 illustrates the nucleotide sequence for c-erg cDNA SEQ ID NO: 4and the deduced amino acid sequence for c-erg protein SEQ ID NO: 4,where the nucleotide sequence lacking the underlined portion of sequencein the figure corresponds to the C-11 gene nucleotide sequence accordingto this invention.

FIG. 2 illustrates the gene domain encoding the c-erg protein, whereETA, NRT, EDB, and CTA represent an erg/ets transcription domain, atranscription repressor domain, an erg/ets DNA-binding domain, and acarboxyl terminal transcription domain, respectively.

FIG. 3 illustrates an autoradiograph of the gel electrophoresis resultsshowing RT-PCR amplified products obtained in Example 2-4, where a 473bp band and a 392 bp band, correspond to c-erg and C-11, respectively.

FIG. 4 illustrates the results comparing DNA synthesizing abilities ofRcas (osteoblasts infected with RCAS only), c-erg-L14 (osteoblastsinfected with a c-erg sense segment-introduced RCAS), and c-erg-L44(osteoblasts infected with a c-erg antisense segment-introduced RCAS).

FIG. 5 illustrates the results comparing the DNA synthesizing abilitiesof Rcas (the osteoblasts infected with RCAS only) and C-11-L14(osteoblasts infected with a C-11 sense segment-introduced RCAS).

FIG. 6 illustrates the results comparing alkaline phosphatase activitiesof Rcas (the osteoblasts infected with only RCAS), c-erg-L14 (theosteoblasts infected with the c-erg sense segment-introduced RCAS) andc-erg-L44 (the osteoblasts infected with the c-erg antisensesegment-introduced RCAS).

FIG. 7 illustrates the results comparing the alkaline phosphataseactivities of Rcas (the osteoblasts infected with RCAS only) andC-11-L14 (the osteoblasts infected with the C-11 sensesegment-introduced RCAS).

FIG. 8 illustrates a photograph showing the results on deposition ofcalcified products in a culture system consisting of uninfectedosteoblasts (the upper half) and the osteoblasts infected with RCAS only(the lower half) as measured by the Alizarin Red staining.

FIG. 9 illustrates a photograph showing the results on deposition ofcalcified products in a culture system consisting of the osteoblastsinfected with c-erg-L14 RCAS (sense segment) (the upper half) and theosteoblasts infected with c-erg-L44 RCAS (antisense segment) (the lowerhalf) as measured by the Alizarin Red staining.

FIG. 10 illustrate a photograph showing the results on deposition ofcalcified products in a culture system consisting of the osteoblastsinfected with RCAS only, the osteoblasts infected with c-erg-L14 RCAS(sense segment), the osteoblasts infected with c-erg-L44 RCAS (antisensesegment), and the osteoblasts infected with C-11-L14 RCAS (sensesegment) as measured by the Alizarin Red staining.

FIG. 11 illustrates a photograph showing the results on deposition ofcalcified products within cell nuclei in a culture system consisting ofthe osteoblasts infected with RCAS only, the osteoblasts infected withc-erg-L14 RCAS (sense segment), the osteoblasts infected with c-erg-L44RCAS (antisense segment), and the osteoblasts infected with C-11-L14RCAS (sense segment) as measured by the von Kossa staining (the righttwo columns) in addition to those obtained by double-staining the cellnuclei of the respective osteoblasts with Alum-carmine (the leftcolumn).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have discovered a novel isoform gene of the c-erggene (herein referred to as “C-11 gene” or “C11 gene”) which is an erggene derived from chicken and determined its nucleotide sequence.Furthermore, the inventors have confirmed the expression of a proteinencoded by such gene (herein referred to as “C-11 protein” or “C11protein”).

Also, the inventors have discovered that when the c-erg or C-11 gene isintroduced into osteoblasts, the calcification of the blasts (or cells)is inhibited.

More specifically, this invention provides a C-11 protein comprising aprotein selected from the group consisting of:

(a) a protein comprising an amino acid sequence having SEQ ID NO. 2(SEQUENCE LISTING), and

(b) a protein comprising an amino acid sequence that is derived from theamino acid sequence having SEQ ID NO. 2 by deletion, substitution orinsertion of one or more amino acids, said protein havingcell-calcification inhibitory activity.

Also, the invention provides a gene encoding the aforementioned protein.

Further, the invention provides a pharmaceutical composition comprisingthe aforementioned protein as well as provides a pharmaceuticalcomposition intended for a cell-calcification inhibitor.

Also, the invention provides a cell-calcification inhibitor comprising ac-erg protein selected from the group consisting of:

(a) a protein comprising an amino acid sequence having SEQ ID NO. 4(SEQUENCE LISTING); and

(b) a protein comprising an amino acid sequence that is derived from theamino acid sequence having SEQ ID NO. 4 by deletion, substitution orinsertion of one or more amino acids, said protein havingcell-calcification inhibitory activity.

Further, the invention provides an antibody to the C-11 protein as wellas provides an antibody, characterized in that said antibody is amonoclonal antibody.

Still further, the invention provides a method for measuring thecalcification of cells comprising the step of measuring the expressionof a C-11 gene or a c-erg gene in the cells.

Also, the invention provides the aforementioned method wherein theexpression of the gene is measured by the amount of C-11 mRNA expressedin the cells or the amount of c-erg mRNA expressed in the cells using aprobe against a DNA sequence specific to the C-11 gene or to the c-erggene.

Also, the invention provides the aforementioned method wherein theexpression of the gene is measured by the amount of expression of theC-11 protein in the cells or the amount of expression of the c-ergprotein in the cells.

Further, the invention provides the aforementioned method wherein theexpression of the C-11 protein or the c-erg protein in the cells ismeasured by means of an antibody to the C-11 protein or to the c-ergprotein.

Also, the invention provides a method for diagnosing such diseases asOPLL (Ossification of posterior longitudinal ligament) andosteoarthritis which cause pathologic calcification or ossification,said method comprising using the aforementioned method of measurement ofthe cell-calcification.

In addition, the invention provides a kit for measuring thecalcification of cells, comprising either or both of an antibody to theC-11 protein and an antibody to the c-erg protein.

Further, the invention provides a method for screening a substancehaving cell-calcification inhibitory blocking activity, said methodcomprising using cells transformed with a gene encoding a proteinselected from the group consisting of:

(a) a protein comprising an amino acid sequence having SEQ ID NO. 2(SEQUENCE LISTING);

(b) a protein comprising an amino acid sequence that is derived from theamino acid sequence having SEQ ID NO. 2 by deletion, substitution orinsertion of one or more amino acids, said protein havingcell-calcification inhibitory activity;

(c) a protein comprising an amino acid sequence having SEQ ID NO. 4(SEQUENCE LISTING); and

(d) a protein comprising an amino acid sequence that is derived from theamino acid sequence having SEQ ID NO. 4 by deletion, substitution orinsertion of one or more amino acids, said protein havingcell-calcification inhibitory activity.

Furthermore, the invention provides a pharmaceutical compositioncomprising an erg protein.

Also, the invention provides a pharmaceutical composition comprising anerg gene.

Further, the invention provides a pharmaceutical composition comprisingthe C-11 protein or the c-erg protein.

In addition, the invention provides a pharmaceutical compositioncomprising the C-11 gene or the c-erg gene.

Still further, the invention provides a pharmaceutical compositioncomprising a protein having a consensus amino acid sequence between thec-erg protein and the C-11 protein.

In the present specification and the accompanying drawings whereabbreviations are used to describe bases and amino acids. Theabbreviations according to the IUPAC-IUB rules or those ascertainable inthe art to which the invention pertains are used as set forth in thefollowing:

Nucleic Acid DNA Deoxyribonucleic acid cDNA Complementary DNA RNARibonucleic acid mRNA Messenger RNA A Adenine C Cytosine G Guanine TThymine

Isolation and Identification of C-11 Gene

The novel gene according to this invention is an isoform of the c-erggene derived from chickens. As shown in FIG. 1, when compared with thenucleotide sequence for the c-erg gene, the novel gene has a nucleotidesequence lacking 81 nucleotides from nucleotide 655 to nucleotide 735(See, SEQ ID NO. 1 in the Sequence Listing). The novel gene according tothe invention (or C-11 gene) can be isolated from a variety of cells bya method ordinarily known in the art.

Specifically, total RNA is extracted from chicken embryos, 4-10 daysold. After the RNA has been subjected to reverse transcription, theentire translational region of the c-erg gene and C-11 gene can beamplified by the PCR method.

Primers that can be used in PCR, for example, include the following C11Band C11C which amplify both c-erg and C-11 genes:

C11B:5′-CACATTATGGCAAGCACTATTAAGG- 3′

C11C:5′-CACTTAGTAGTAGGTGCCAAGATGG- 3′

C11A:5′-ATCTTGATCACATTATGGCAAGC- 3′

When Primers C11B and C11C are each used, two bands, 1446 bp and 1355bp, appear: the former band corresponds to the c-erg gene and the latterband to the C-11 gene, respectively. When Primers C11A and C11C are eachused, two kinds of DNAs, 1454 bp (c-erg) and 1373 bp (C-11), can beamplified.

In either case, the RT-PCR conditions are as follows: 1 μg of total RNAis subjected to reverse transcription and subsequently to 30 cycles ofamplification for one minute at 61° C., for two minutes at 72° C. andfor 10 seconds at 95° C. employing 2 units of Tag polymerase to renderthe products detectable.

In addition, based on the determined nucleotide sequence as describedabove it is easy to prepare an oligonucleotide probe complementary to anappropriate partial nucleotide sequence thereof. Such probes may be usedto enable detection of the C-11 gene by various techniques.

C-11 Protein

The C-11 protein which can be deduced from the nucleotide sequence forthe C-11 gene has an amino acid sequence set forth in SEQ ID NO. 2 inthe Sequence Listing.

The C-11 protein can also be expressed in suitable host cells byincorporating the C-11 gene into viral vectors such as adenovirus andchicken retrovirus. See Gene Transfer and Expression: A LaboratoryManual by Michael Kriegler, W.H. Freeman and Company, New York (1991),p29-56. Further, the amino acid sequence of the proteins thus obtainedcan be directly determined on a conventional amino acid sequencer.

Furthermore, it is possible to modify the sequence of the C-11 gene setforth as SEQ ID NO. 1 in the Sequence Listing by substitution, deletionor insertion of an arbitrary sequence in said sequence using, forexample, site-directed mutagenesis. The site-directed mutagenesis can berepeated one to a few times to prepare C-11 protein variants in whichone to a few amino acids are substituted, deleted or inserted as well asgene mutants encoding the variants. Such variants are within the scopeof the invention insofar as they possess cell-calcification inhibitoryactivity.

Also, various methods known in the art may be used to detect the C-11protein expressed within particular cells. Specifically, representativemethods include immunostaining using an antibody to the C-11 protein ora partial protein thereof, a method for the detection of theirlocalization by the use of a fluorescent antibody technique, and amethod for measuring the amount of expression of the C-11 protein bymeans of a radioimmunoassay or ELISA technique on homogenated cells.

c-erg Protein

In a manner similar to that used for the C-11 protein, it is possible toexpress the c-erg protein set forth as SEQ ID NO. 4 in the SequenceListing.

Further, variants of the c-erg protein or mutants of their genes can beprepared in a manner similar to that used for the C-11 protein or genesthereof, and can satisfactorily be utilized in this invention insofar asthey possess cell-calcification inhibitory activity.

Antibody (Monoclonal Antibody) to C-11 Protein

An antibody to the C-11 protein according to the invention may be thatwhich reacts against either the aforementioned entire C-11protein or apartial oligopeptide thereof. In addition, it is possible to use anantibody conjugated with a suitable substance such as a protein whichwill impart particular properties to the antibody.

There is no limitation concerning immunological methods in which theaforementioned proteins etc., are used as antigens, and anyimmunological techniques ordinarily known in the art may be used.Following such methods, a serum containing polyclonal antibodies may beobtained. Further, particular fractions such as IgG can be obtained bypurification through ammonium sulfate fractionation or on Protein ASepharose.

Furthermore, it is also possible to prepare monoclonal antibodies by thecell fusion method.

Cell-calcification Inhibitory Activity

“Cell-calcification inhibitory activity” caused by introduction of theC-11 gene or the c-erg gene according to the invention, into cells meansthe inhibition of calcification inductive ability by osteoblastsinfected with viral vectors having the aforementioned genes cloned. Inthis invention the Alizarin Red method may preferably be used to measurethe amount of deposition of calcified products which the cells haveinduced. Also, the von Kossa method (also known as the Alum-carminemethod) permits easy discrimination.

Pharmaceutical Compositions and Methods for their Administration

The pharmaceutical compositions according to the invention arecharacterized by containing the erg protein, erg gene, C-11 protein,c-erg protein, C-11 gene or c-erg gene, each of which has the functionto inhibit the calcification of cells as described above. Accordingly,by virtue of the cell-calcification inhibition, those compositions arecapable of treating various diseases, more specifically those in whichpathological calcification causes ossification such as OPLL andosteoarthritis.

The pharmaceutical compositions provided by the invention are thosewhich contain the aforementioned erg protein, erg gene, C-11 protein,c-erg protein, C-11 gene or c-erg gene. Method for administration ofthose pharmaceutical compositions are not particularly limited, andconventional administration method are available for use. Specifically,these include local injection, subcutaneous injection and oraladministration. In addition, intracellular microinjection or the likemay be indicated.

More specifically, by introducing into the cells, the c-erg protein orthe C-11 protein in its form bound to a suitable hormone or the like, orin its form as a fused protein, it is possible to have the c-erg or C-11protein bound to receptors for the hormone, which allows the c-erg orC-11 protein together with the hormone to be taken up within the cells.

EXAMPLE 1

Isolation of C-11 Gene and c-erg Gene

Sterna were separated from chicken embryos, 18 days old and total RNAwas then extracted. See, Iwamoto M. et al., Microscopy Research andTechnique (1994) 28: 483-491. After the RNA thus obtained had beensubjected to reverse transcription, amplification was allowed to proceed30 cycles for one minute at 61° C., for two minutes at 72° C., and for10 seconds at 95° C., respectively using PCR Primers (C11A and C11C, orC11B and C11C) which amplify the entire coding region for the c-erggene. With either pair of the primers, two bands were obtained.Respective bands were cut out and DNA fragments were identified using aQiaexII gel extraction kit (Qiagen, Germany). These fragments weresubcloned into a PCRII vector (Invitrogen, CA, USA), and then thefull-length nucleotide sequences were determined by the Dideoxy method.As a result, a 1454 bp DNA fragment amplified with the primer pair ofC11A and C11C completely matched the c-erg gene (Mechanism ofDevelopment (1995) 50: 17-28), whereas a 1373 bp DNA fragment turned outto be a sequence lacking 81 bases of from nucleotide 655 to nucleotide735, from the c-erg gene. This novel sequence thus obtained wasdesignated “C-11”.

DNA fragments amplified using the primer pair of C11B and C11C were twokinds, namely 1446 bp and 1335 bp: the former fragment was c-erg and thelatter was C-11. These results suggest the possibility that the thusobtained C-11 gene is an isoform of the c-erg gene.

It was also possible to isolate the aforementioned C-11 gene by aconventional subtraction method. Particularly, according to thesubtraction method ordinarily known in the art, a cDNA which hadspecifically expressed in the embryonic sterna was cloned by employing acDNA library of chicken embryonic sterna and fibroblasts. From the thusobtained candidate clones, genes were amplified by the PCR reactionusing suitable primers to provide amplified gene products, the fullnucleotide sequences of which were determined by standard techniques.

Results obtained with the aforementioned c-erg gene are shown throughExample 2-1 to Example 2-8 as described below. In a like manner,experiments were conducted using the aforementioned C-11 gene andresults therefrom are shown below, together with these for the c-erggene.

EXAMPLE 2-1

Construction of a Vector Containing the c-erg Gene

The cloned c-erg as described above was subcloned into a RCAS vector(See, Journal of Virology (1987), October: 3004-3012) at its ClaI sitein sense (c-erg) and antisense (AS-c-erg) directions, respectively. Thatthey were indeed sense and antisense as described was confirmed by DNAsequencing.

The RCAS vector containing the subcloned c-erg and that containing thesubcloned AS-c-erg were, respectively, introduced into chicken embryonicfibroblasts by the calcium phosphate co-precipitation method asdescribed in Chen C. and Okayama H., Mol. Cell Biol. (1987), 7:2745-2752. The vector-introduced cells were cultured for 48 hours at 37°C. on a DMEM medium containing 10% fetal bovine serum (The NikkenBiological Science Research Institute, Kyoto). Subsequently, virusproduced in the culture supernatant was concentrated by means of aultrafiltration membrane (molecular weight: 30,000 cut) (Centriprepavailable from Amicon Inc., Mass., USA). This virus is herein referredto as “virus-CM”.

In a like manner, only the RCAS vector was introduced into chickenembryonic fibroblasts and the virus recovered from the vector-introducedcells was used as a control.

A virus stock from the aforementioned chicken embryonic fibroblastswhich was used in this invention was prepared by the method as describedbelow. Specifically, CEF was transfected with a DNA which had beenobtained from RSV (Rous Sarcoma Virus) by substitution of its v-src witha target gene by means of Calcium phosphate method and which contained aproviral structure with LTR at its both ends. The cells into which thegenes were introduced, temporarily released a large quantity of virus.Then, the virus reached the state of propagation throughout the wholeculture used in the transfection by virus infection. At this point, avirus stock was recovered from the culture medium. More specifically,CEF cells propagated in a confluent manner, which had been cultured 4-5days after preparation, were inoculated in a 60 mm dish at 0.8×10⁶cells. On the following day, 10 μg of a DNA fragment or plasmid, each ofwhich had been provided with a proviral structure, was introduced intothe cells by transfection, according to the calcium phosphateco-precipitation method. The medium was made afresh one to two hoursprior to transfection. Ten hours after transfection, the culture mediumwas washed three times with a standard medium and then the cells wereallowed to propagate for two days. The whole culture transfected waspassaged to a 90 mm dish and allowed to propagate for two additionaldays. The medium was made afresh and it was recovered as the virus stockafter 48 hours. A new medium was again added to the culture medium and,after 12 hours, a virus stock was recovered for the second time.

EXAMPLE 2—2

Introduction of the c-erg Gene into Cells

The c-erg gene was introduced into chicken osteoblasts to observe theeffects according to the method of Iwamoto et al. as described in J.Biol. Chem., (1993) 268(13): 9645-52. Parietal bones were excised from achicken, 18 days old and osteoblasts for use were isolated from thebones by the method of Louis C.G. et al. as described in DevelopmentalBiology (1987), 122: 49-60.

Virus-CM (and the control virus) prepared in Example 2-1 was added tothe osteoblasts prepared as described above and virus infection wasallowed to take place.

Whether or not the infection had occurred was determined by observingchanges in the differentiation character of the cells. Infectionefficiency toward virus cells was determined by staining the osteoblastsusing a P19 antivirus antibody (Development Studies Hybridoma Bank) asdescribed in Potts W. M. et al., J. Gen. Viol., (1982), 68: 3177-3182.

EXAMPLE 2-3

Morphological Observation of a Transformant with the c-erg Gene

The virus-infected osteoblasts obtained in Example 2—2 were observedunder a phase contrast microscope with a magnification of 10. The resultrevealed that when compared with the cells in the control group, thevirus-infected osteoblasts showed polygonal morphology in a smaller size(data not shown in the Drawings).

EXAMPLE 2-4

Detection of Expression of the c-erg Gene and C-11 Gene

To detect the expression of the aforementioned isoform, the C-11 gene,the RT-PCR method was performed according to the procedure as describedbelow.

Total RNA was prepared from the pectus spinal tissue of chicken embryos,18 days old by the method of Iwamoto et al. as described in MicroscopyResearch and Technique (1994), 28: 483-491. After converting this RNAinto a DNA by means of a random hexamer and a superscript reversetranscriptase (both available from Gibco-BRL, MD, USA), the PCRamplification reaction was performed using Primers C11A and C11C (SEQ IDNO. 5 and SE ID NO. 7 set forth in the Sequence Listing) which had thenucleotide sequence for the ETA region (erg/ets transcription region) ofthe c-erg gene shown in FIG. 2, and that for the NRT region(transcription repressor region), respectively. In FIG. 2, the one thatlacks 81 bases being flanked with the ETA and NRT domains is the C-11gene. The thus obtained amplified products were subjected toelectrophoresis on a 2% agarose gel and the results are shown in FIG. 3.These results revealed that the kinds of mRNA were amplified to show theisoform G11 gene as a 392 bp band together with the c-erg gene (as a 473bp band).

EXAMPLE 2-5

Measurement of DNA Synthesizing Ability

The DNA synthesizing ability of the c-erg transformant cells wasmeasured by the procedure described below.

After washing the cells with a cooled physiological saline solutionthree times, the cells were recovered from a physiological salinesolution containing 0.01N NaOH and 0.2 v/v % Triton X-100. Uponrecovery, the cells were crushed by ultrasonication and centrifuged toprovide a supernatant which was used as a sample in the measurement. To100 μl supernatant was added 200 μl of 0.1 g/ml DABA (3,5-diaminobenzoicacid dihydrochloride). After incubation for 45 minutes at 65° C. under alight-shielding condition, the reaction was terminated by adding 300 μlof 2N HCl. Upon termination of the reaction, fluorescence was monitoredat wavelengths of 420 and 510 nm.

As used herein, “Rcas (or RCAS)” means the cells infected with thevector only. “c-erg-L14” means a RCAS into which a sense segment of thec-erg gene has been introduced. “c-erg-L44” means a RCAS into which anantisense segment of the c-erg gene has been introduced. Further,“C-11-L14” means a RCAS into which a sense segment of the C-11 gene hasbeen introduced.

As shown in FIG. 4, it was found that either of c-erg-L14 and c-erg-L44(in-to both of which c-erg had been introduced) showed no significantdifference in the DNA synthesizing ability as compared with RCAS (intowhich c-erg had not been introduced). In contrast, as shown in FIG. 5,it was found that C-11-L14 (into which C-11 had been introduced) showeda significant increase in the DNA synthesizing ability as compared withRCAS itself. This increase was particularly noted until day 6.

EXAMPLE 2-6

Measurement of Alkaline Phosphatase Activity

The alkaline phosphatase activity was measured according to the methodof Kato Y. et al. as described in Endocrimology (1990), 127: 114-118.

Specifically, after washing the cells on ice with a cooled physiologicalsaline solution three times, the cells were recovered from aphysiological saline solution containing 0.2 v/v % Triton X-100. Uponrecovery, the cells were crushed by ultrasonication and centrifuged toprovide a supernatant which was used as a sample in the measurement.

The alkaline phosphatase activity of the supernatant was measured in0.5M Tris/HCl buffer (pH 9.0) containing 0.5 mM pNP (para-nitrophenylphosphate) and 0.5 mM MgCl₂. After incubating the mixed solution for 30minutes at 37° C., the reaction was terminated by adding 0.25 volume of1N NaOH. Upon termination of the reaction, absorbance was monitored at awavelength of 410 nm.

The results obtained employing RCAS, into which c-erg was introduced,are shown in FIG. 6. While no significant difference was observedbetween RCAS (which was the control) and c-erg-L44 (anti-sense segmentintroduced), the alkaline phosphatase activity of c-erg-L14(sense-segment introduced) was found to be markedly inhibited.

Similarly, the results obtained employing C-11-L14, into which a sensesegment of the C-11 gene was introduced, are shown in FIG. 7. Ascompared with RCAS which was the control, the alkaline phosphataseactivity of C-11-L14 was found to be markedly inhibited.

EXAMPLE 2-7

Alizarin Red Staining

After washing the cultured osteoblasts (on day 12 after inoculation)with PBS (phosphate buffer saline) twice the cells were fixed with 100%ethanol. After fixation, Alizarin Red S (sodium alizarin sulfonateavailable from Wako Pure Chemicals) was dissolved in distilled water andadjusted to pH 6.3-6.4 with 0.1 N NH₃, yielding a 1% Alizarin solution.This was added to the cells to effect staining for two minutes. Afterstaining, the cells were washed with distilled water and air-dried.

Following the abovementioned operations, the calcification sites of thecells were stained reddish orange.

FIG. 8 illustrates the results obtained by measuring the amount ofdeposition of calcified products in a culture system of the osteoblastson day 19 after inoculation by means of Alizarin Red. Both uninfectedcells and cells infected with the RCAS vector itself showed similardegrees of deposition of the calcified products. On the other hand, forc-erg-L14 (c-erg sense segment-introduced RCAS), the deposition wasabout half that for c-erg-L44 (c-erg anti-sense segment-introducedRCAS).

Further, FIG. 9 illustrates that in the case of a C-11-L14 RCAS intowhich a sense segment of C-11 was introduced, almost no deposition ofthe calcified products was observed as the result of measurement withAlizarin Red.

FIG. 10 illustrates the results from staining with Alizarin Red tocompare the amounts of deposition of such calcified products. Whencompared under conditions where deposition of the calcified products wasclearly observed in the cells infected with RCAS itself, c-erg L44showed an extremely small inhibition on deposition of the calcifiedproducts. In contrast c-erg-L14 was found to nearly inhibit thedeposition and C-11-L14 was found to almost completely inhibit thedeposition.

EXAMPLE 2-8

von Kossa Staining

After washing the cultured osteoblasts (on day 12 after inoculation)with PBS twice, the cells were fixed with 100% ethanol. Then a 1%solution of silver nitrate was added to the cells and the solution wasexposed to the sunlight for 30 minutes. Subsequently, the cells werewashed with distilled water, to which a 5% aqueous solution of sodiumthiosulfate was added. After allowing to stand for 5-10 minutes, thecells were washed with water. After washing, staining of cell nuclei wasperformed by adding an Alum-carmine solution and allowing to stand for24 hours.

Following the abovementioned operations, the calcification sites of thecells were stained black, while the nuclei were stained crimson.

As in the Alizarin Red staining, with application of the von Kossastaining a lowered staining was observed and the deposition of thecalcified products was inhibited in the c-erg-L14 group as compared withthe control group (RCAS). Further, in the C-11-L14 group, the depositionwas completely inhibited. The staining with Alum-carmine showed nodiscrimination among various groups. Therefore, the observed effects inthis invention are not ascribable to variations in staining efficiencyresulting from differences in the cell number, but are believed to bedue to the fact that both c-erg-L14 and C11-L14 functionally inhibit thecalcification of the osteoblasts (amount of calcium deposited per cell).

7 1 1447 DNA C-11 gene, c-erg gene w/ deletion, chicken DNA 1 gaattccgcgaacgaataat tattattagc aattattagc gatcaataat cttgatcaca 60 ttatggcaagcactattaag gaagcattat cagtggtgag tgaagaccag tccttgtttg 120 agtgtgcctacggatcgccc caccttgcaa agacagaaat gacagcctcc tcttccagtg 180 aatatgggcaaacatcaaag atgagcccgc gcgttcccca gcaggactgg ttatcacagc 240 ccccggccagagttaccatt aagatggagt gtaacccaaa ccaggttaat gggtcaagga 300 attcacctgatgactgcagc gtggcaaaag gagggaaaat ggttagcagt tcagacaatg 360 ttgggatgaactatggaagc tacatggaag agaagcatat tccgcctcca aatatgacaa 420 ccaatgaacgaagagttatt gtgccagcag atcctacgtt atggagcaca gaccatgtac 480 ggcagtggctggagtgggca gtgaaggagt atggtcttcc agacgtggac atcttgttgt 540 tccagaacattgatgggaaa gagttgtgta aaatgaccaa agatgacttc cagagactca 600 cgccgagctataacgcagat atcctcctgt cacacctaca ctacctcaga gagagaggag 660 ccacttttatttttccaaat acatcagttt acccagaagc aacgcaaaga ataacaacaa 720 ggccagatttaccttatgag caagcgagga gatcagcgtg gacgagtcac agccatccca 780 ctcagtcaaaagctacccaa ccatcatctt caacagtgcc caaaacagaa gaccagcgtc 840 ctcagttagatccttatcag attcttggac cgaccagcag ccgtcttgca aatccaggga 900 gtgggcagatacagctatgg cagttcctac tggagcttct gtcggacagc tccaactcca 960 actgcatcacctgggagggc acaaatgggg agttcaagat gacagaccct gatgaagtgg 1020 ctcggcgttggggagagagg aaaagcaaac ctaacatgaa ctatgacaaa ctcagccgtg 1080 cacttcgctactactatgac aaaaatatta tgactaaagt tcatggtaaa cgctatgcct 1140 acaaatttgatttccacgga atcgctcagg ccctccagcc tcaccctcca gaatcatcca 1200 tgtacaaatacccatcagac ctcccctaca tgagttccta ccatgcacac ccccagaaga 1260 tgaactttgtagctccccat ccccctgctt tgcccgtaac ctcatccagc ttttttgctg 1320 cccctaatccatactggaat tcaccaactg gaggcatcta ccccaatacc aggctgccag 1380 ctgctcatatgccttcccat cttggcacct actactaagt ggggaaagaa agaaagcgcc 1440 aagaaaa 14472 451 PRT protein sequence from C-11 gene 2 Met Ala Ser Thr Ile Lys GluAla Leu Ser Val Val Ser Glu Asp Gln 1 5 10 15 Ser Leu Phe Glu Cys AlaTyr Gly Ser Pro His Leu Ala Lys Thr Glu 20 25 30 Met Thr Ala Ser Ser SerSer Glu Tyr Gly Gln Thr Ser Lys Met Ser 35 40 45 Pro Arg Val Pro Gln GlnAsp Trp Leu Ser Gln Pro Pro Ala Arg Val 50 55 60 Thr Ile Lys Met Glu CysAsn Pro Asn Gln Val Asn Gly Ser Arg Asn 65 70 75 80 Ser Pro Asp Asp CysSer Val Ala Lys Gly Gly Lys Met Val Ser Ser 85 90 95 Ser Asp Asn Val GlyMet Asn Tyr Gly Ser Tyr Met Glu Glu Lys His 100 105 110 Ile Pro Pro ProAsn Met Thr Thr Asn Glu Arg Arg Val Ile Val Pro 115 120 125 Ala Asp ProThr Leu Trp Ser Thr Asp His Val Arg Gln Trp Leu Glu 130 135 140 Trp AlaVal Lys Glu Tyr Gly Leu Pro Asp Val Asp Ile Leu Leu Phe 145 150 155 160Gln Asn Ile Asp Gly Lys Glu Leu Cys Lys Met Thr Lys Asp Asp Phe 165 170175 Gln Arg Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu 180185 190 His Tyr Leu Arg Glu Arg Gly Ala Thr Phe Ile Phe Pro Asn Thr Ser195 200 205 Val Tyr Pro Glu Ala Thr Gln Arg Ile Thr Thr Arg Pro Asp LeuPro 210 215 220 Tyr Glu Gln Ala Arg Arg Ser Ala Trp Thr Ser His Ser HisPro Thr 225 230 235 240 Gln Ser Lys Ala Thr Gln Pro Ser Ser Ser Thr ValPro Lys Thr Glu 245 250 255 Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln IleLeu Gly Pro Thr Ser 260 265 270 Ser Arg Leu Ala Asn Pro Gly Ser Gly GlnIle Gln Leu Trp Gln Phe 275 280 285 Leu Leu Glu Leu Leu Ser Asp Ser SerAsn Ser Asn Cys Ile Thr Trp 290 295 300 Glu Gly Thr Asn Gly Glu Phe LysMet Thr Asp Pro Asp Glu Val Ala 305 310 315 320 Arg Arg Trp Gly Glu ArgLys Ser Lys Pro Asn Met Asn Tyr Asp Lys 325 330 335 Leu Ser Arg Ala LeuArg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys 340 345 350 Val His Gly LysArg Tyr Ala Tyr Lys Phe Asp Phe His Gly Ile Ala 355 360 365 Gln Ala LeuGln Pro His Pro Pro Glu Ser Ser Met Tyr Lys Tyr Pro 370 375 380 Ser AspLeu Pro Tyr Met Ser Ser Tyr His Ala His Pro Gln Lys Met 385 390 395 400Asn Phe Val Ala Pro His Pro Pro Ala Leu Pro Val Thr Ser Ser Ser 405 410415 Phe Phe Ala Ala Pro Asn Pro Tyr Trp Asn Ser Pro Thr Gly Gly Ile 420425 430 Tyr Pro Asn Thr Arg Leu Pro Ala Ala His Met Pro Ser His Leu Gly435 440 445 Thr Tyr Tyr 450 3 1528 DNA c-erg gene, chicken DNA 3gaattccgcg aacgaataat tattattagc aattattagc gatcaataat cttgatcaca 60ttatggcaag cactattaag gaagcattat cagtggtgag tgaagaccag tccttgtttg 120agtgtgccta cggatcgccc caccttgcaa agacagaaat gacagcctcc tcttccagtg 180aatatgggca aacatcaaag atgagcccgc gcgttcccca gcaggactgg ttatcacagc 240ccccggccag agttaccatt aagatggagt gtaacccaaa ccaggttaat gggtcaagga 300attcacctga tgactgcagc gtggcaaaag gagggaaaat ggttagcagt tcagacaatg 360ttgggatgaa ctatggaagc tacatggaag agaagcatat tccgcctcca aatatgacaa 420ccaatgaacg aagagttatt gtgccagcag atcctacgtt atggagcaca gaccatgtac 480ggcagtggct ggagtgggca gtgaaggagt atggtcttcc agacgtggac atcttgttgt 540tccagaacat tgatgggaaa gagttgtgta aaatgaccaa agatgacttc cagagactca 600cgccgagcta taacgcagat atcctcctgt cacacctaca ctacctcaga gagactcctc 660ttccacattt gacttcagat gatgttgata aggccttaca aaactctcca cggttaatgc 720atgctagaaa cacaggagga gccactttta tttttccaaa tacatcagtt tacccagaag 780caacgcaaag aataacaaca aggccagatt taccttatga gcaagcgagg agatcagcgt 840ggacgagtca cagccatccc actcagtcaa aagctaccca accatcatct tcaacagtgc 900ccaaaacaga agaccagcgt cctcagttag atccttatca gattcttgga ccgaccagca 960gccgtcttgc aaatccaggg agtgggcaga tacagctatg gcagttccta ctggagcttc 1020tgtcggacag ctccaactcc aactgcatca cctgggaggg cacaaatggg gagttcaaga 1080tgacagaccc tgatgaagtg gctcggcgtt ggggagagag gaaaagcaaa cctaacatga 1140actatgacaa actcagccgt gcacttcgct actactatga caaaaatatt atgactaaag 1200ttcatggtaa acgctatgcc tacaaatttg atttccacgg aatcgctcag gccctccagc 1260ctcaccctcc agaatcatcc atgtacaaat acccatcaga cctcccctac atgagttcct 1320accatgcaca cccccagaag atgaactttg tagctcccca tccccctgct ttgcccgtaa 1380cctcatccag cttttttgct gcccctaatc catactggaa ttcaccaact ggaggcatct 1440accccaatac caggctgcca gctgctcata tgccttccca tcttggcacc tactactaag 1500tggggaaaga aagaaagcgc caagaaaa 1528 4 478 PRT protein sequence fromc-erg gene 4 Met Ala Ser Thr Ile Lys Glu Ala Leu Ser Val Val Ser Glu AspGln 1 5 10 15 Ser Leu Phe Glu Cys Ala Tyr Gly Ser Pro His Leu Ala LysThr Glu 20 25 30 Met Thr Ala Ser Ser Ser Ser Glu Tyr Gly Gln Thr Ser LysMet Ser 35 40 45 Pro Arg Val Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro AlaArg Val 50 55 60 Thr Ile Lys Met Glu Cys Asn Pro Asn Gln Val Asn Gly SerArg Asn 65 70 75 80 Ser Pro Asp Asp Cys Ser Val Ala Lys Gly Gly Lys MetVal Ser Ser 85 90 95 Ser Asp Asn Val Gly Met Asn Tyr Gly Ser Tyr Met GluGlu Lys His 100 105 110 Ile Pro Pro Pro Asn Met Thr Thr Asn Glu Arg ArgVal Ile Val Pro 115 120 125 Ala Asp Pro Thr Leu Trp Ser Thr Asp His ValArg Gln Trp Leu Glu 130 135 140 Trp Ala Val Lys Glu Tyr Gly Leu Pro AspVal Asp Ile Leu Leu Phe 145 150 155 160 Gln Asn Ile Asp Gly Lys Glu LeuCys Lys Met Thr Lys Asp Asp Phe 165 170 175 Gln Arg Leu Thr Pro Ser TyrAsn Ala Asp Ile Leu Leu Ser His Leu 180 185 190 His Tyr Leu Arg Glu ThrPro Leu Pro His Leu Thr Ser Asp Asp Val 195 200 205 Asp Lys Ala Leu GlnAsn Ser Pro Arg Leu Met His Ala Arg Asn Thr 210 215 220 Gly Gly Ala ThrPhe Ile Phe Pro Asn Thr Ser Val Tyr Pro Glu Ala 225 230 235 240 Thr GlnArg Ile Thr Thr Arg Pro Asp Leu Pro Tyr Glu Gln Ala Arg 245 250 255 ArgSer Ala Trp Thr Ser His Ser His Pro Thr Gln Ser Lys Ala Thr 260 265 270Gln Pro Ser Ser Ser Thr Val Pro Lys Thr Glu Asp Gln Arg Pro Gln 275 280285 Leu Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg Leu Ala Asn 290295 300 Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu Leu305 310 315 320 Ser Asp Ser Ser Asn Ser Asn Cys Ile Thr Trp Glu Gly ThrAsn Gly 325 330 335 Glu Phe Lys Met Thr Asp Pro Asp Glu Val Ala Arg ArgTrp Gly Glu 340 345 350 Arg Lys Ser Lys Pro Asn Met Asn Tyr Asp Lys LeuSer Arg Ala Leu 355 360 365 Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr LysVal His Pro Pro Glu 370 375 380 Ser Ser Met Tyr Lys Tyr Pro Ser Asp LeuPro Tyr Met Ser Ser Tyr 385 390 395 400 His Gly Lys Arg Tyr Ala Tyr LysPhe Asp Phe His Gly Ile Ala Gln 405 410 415 Ala Leu Gln Pro His Ala HisPro Gln Lys Met Asn Phe Val Ala Pro 420 425 430 His Pro Pro Ala Leu ProVal Thr Ser Ser Ser Phe Phe Ala Ala Pro 435 440 445 Asn Pro Tyr Trp AsnSer Pro Thr Gly Gly Ile Tyr Pro Asn Thr Arg 450 455 460 Leu Pro Ala AlaHis Met Pro Ser His Leu Gly Thr Tyr Tyr 465 470 475 5 23 DNA primer forisolation of C-11 and c-erg genes 5 atcttgatca cattatggca agc 23 6 25DNA primer for isolation of C-11 and c-erg genes 6 cacattatgg caagcactattaagg 25 7 25 DNA primer for isolation of C-11 and c-erg genes 7cacttagtag taggtgccaa gatgg 25

What is claimed is:
 1. An isolated nucleic acid encoding a C11 proteincomprising the amino acids as set forth in SEQ ID NO:
 2. 2. A nucleicacid comprising the nucleotide sequence as set forth in SEQ ID NO:
 1. 3.A vector incorporating the isolated nucleic acid of claim 1 or
 2. 4. Thevector of claim 3, wherein said vector is an expression vector.
 5. Ahost cell comprising the vector of claim
 3. 6. A host cell comprisingthe vector of claim
 4. 7. The host cell of claim 6, wherein said hostcell is selected from the group consisting of eukaryotic cells andprokaryotic cells.
 8. The host cell of claim 7, wherein said host cellis eukaryotic.
 9. A method of expressing and recovering a protein havingcell calcification inhibitory activity comprising the steps of: (i)transfecting a cell with the vector of claim 3; (ii) propagating saidtransfected cell; and (iii) recovering said protein from said propagatedcells.